G-protein coupled receptors (GPCRs) are proteins responsible for transducing a signal within a cell. GPCRs have usually seven transmembrane domains. Upon binding of a ligand to an extra-cellular portion or fragment of a GPCR, a signal is transduced within the cell that results in a change in a biological or physiological property or behaviour of the cell. GPCRs, along with G-proteins and effectors (intracellular enzymes and channels modulated by G-proteins), are the components of a modular signalling system that connects the state of intra-cellular second messengers to extra-cellular inputs.
GPCR genes and gene products can modulate various physiological processes and are potential causative agents of disease. The GPCRs seem to be of critical importance to both the central nervous system and peripheral physiological processes.
The GPCR protein superfamily is represented by five families: Family I, receptors typified by rhodopsin and the beta2-adrenergic receptor and currently represented by over 200 unique members; Family II, the parathyroid hormone/calcitonin/secretin receptor family; Family III, the metabotropic glutamate receptor family, Family IV, the CAMP receptor family, important in the chemotaxis and development of D. discoideum; and Family V, the fungal mating pheromone receptor such as STE2.
G proteins represent a family of heterotrimeric proteins composed of α, β and γ subunits, that bind guanine nucleotides. These proteins are usually linked to cell surface receptors (receptors containing seven transmembrane domains) for signal transduction. Indeed, following ligand binding to the GPCR, a conformational change is transmitted to the G protein, which causes the α-subunit to exchange a bound GDP molecule for a GTP molecule and to dissociate from the βγ-subunits.
The GTP-bound form of the α, β and γ-subunits typically functions as an effector-modulating moiety, leading to the production of second messengers, such as cAMP (e.g. by activation of adenyl cyclase), diacylglycerol or inositol phosphates.
More than 20 different types of α-subunits are known in humans. These subunits associate with a small pool of β and γ subunits. Examples of mammalian G proteins include Gi, Go, Gq, Gs and Gt. G proteins are described extensively in Lodish et al., Molecular Cell Biology (Scientific American Books Inc., New York, N.Y., 1995; and also by Downes and Gautam, 1999, The G-Protein Subunit Gene Families. Genomics 62:544-552), the contents of both of which are incorporated herein by reference.
Known and uncharacterized GPCRs currently constitute major targets for drug action and development. There are ongoing efforts to identify new G protein coupled receptors which can be used to screen for new agonists and antagonists having potential prophylactic and therapeutic properties.
More than 300 GPCRs have been cloned to date, excluding the family of olfactory receptors. Mechanistically, approximately 50-60% of all clinically relevant drugs act by modulating the functions of various GPCRs (Cudermann et al., J. Mol. Med., 73:51-63, 1995).
GPR72 initially referred to as GIR for glucocorticoid-induced receptor gene is also called JP05 or GPR83 (SEQ ID NO: 1, human polynucleotide sequence, SEQ ID NO: 2 human amino acid sequence). The predicted amino acid sequence was found to share significant similarity with the rhodopsin like G-protein coupled receptors family. The highest homology of GPR72 with known receptors is found with tachykinin receptors NK-1, NK-2, and NK-3 (32, 31 and 33%, respectively). The genomic organization of the mouse GPR72 gene has been determined and compared with the human gene [De Moerlooze et al., Cell Genet. 90 (2000) 146-150]. It is similar in both species, although differences leading to specific splicing variants in the mouse have been found. Comparative genetic mapping of the GPR72 gene showed that it maps to regions of conserved syngeny on mouse chromosome 9 (A2-3 region) and human chromosome 11 (q21 region) [Parker et al. Biochimica and Biophysica Acta, 1491 (2000) 369-375]. The human GPR72 polypeptide shares 89.5% identity with its mouse ortholog (SEQ ID NO:3, mouse polynucleotide sequence, SEQ ID NO:4 mouse amino acid sequence). GPR72 was originally identified as a stress-response element from murine thymoma WEHI-7TG cells after being treated with glucocorticoids and forskolin [Harrigan et al. Mol. Cell. Biol. 9 (1989) 3438-3446; Harrigan et al. Mol. Endocrinol. 5 (1991) 1331-1338]. CNS regulation of GPR72 mRNA following in vivo administration of dexamethasone suggests a potential role of this receptor in glucocorticoid-mediated effects such as, hypothalamic pituitary adrenal (HPA) function and stress regulation [Adams et al. Molecular Brain Research 117 (2003) 39-46]. In addition GPR72 transcript levels are increased significantly in rat prefrontal cortex for 7 days after discontinuation of chronic amphetamine exposure. The induction of GPR72 expression by amphetamine is associated with augmented behavioral activation suggesting that modulation of GPR72 expression may be involved in behavioral sensitization, and GPR72 may play a role at the interface between stress and neuroadaptation to psychostimulants [Wang et al. The journal of neuroscience 21 (2001) 9027-9035]. GPR72 mRNA were detected in high levels in human, rat and murine brain and spinal cord by Northern blot or RT-PCR analysis [Sah et al. Neuroscience 133 (2005) 281-292; Brezillon et al. Brain research 921 (2001) 21-30; Pesini et al. Molecular brain research 57 (1998) 281-300]. More specifically, distribution of GPR72 mRNA was examined in the human forebrain using in situ hybridization analysis. The results revealed a wide but discrete distribution of the transcript with strongly GPR72 mRNA expressing cells, presumably neurons, present in the cerebral cortex (layer II), hippocampus (pyramidal CA3 neurons and granule cells), amygdala (basal and periamygdaloid cortical nuclei), in the endopiriform nucleus, diagonal band of Broca, thalamus (nucleus reuniens, parafascicular nucleus) and hypothalamus (posterior, dorsal, and around the medial mammillary). Weaker signals were detected in the deeper cortical layers and throughout the striatum. A few positive cells were evident in the raphe but not in the substantia nigra or pontine nuclei [Brezillon et al. Brain research 921 (2001) 21-30]. The distribution patterns of GPR72 mRNA in the human brain suggest involvement in control of emotions and of neuroendocrine, cognitive and motor functions.
Polyunsaturated Fatty Acids (PUFAs) are fatty acids containing at least 16 carbons and two or more double bonds, optionally cyclic or branched, and optionally substituted with hydroxyl groups. Some examples are: linolenic acid (LA) (18:2n-6), alpha-linolenic acid (ALA) (18:3n-3), gamma-linolenic acid (GLA) (18:3n-6), arachidonic acid (AA) (20:4n-6), eicosapentaenoic acid (EPA) (20:5n-3), docosahexaenoic acid (DHA) (22:6n-3).
PUFAs occur throughout animal, plant, algae, fungi and bacteria. Found widely in many lipid compounds such as membranes, storage oils, glycolipids, phospholipids, sphingolipids and lipoproteins. Interest in PUFAs arises from their potential in therapeutic applications as well as in food and nutritional applications. They are produced commercially from selected seed plants, and some marine sources.
PUFAs provide structural and functional characteristics, and are involved in a wide range of biological components including membranes (in phospholipids). They are involved in regulating architecture, dynamics, phase transitions and permeability of membranes, and control of membrane-associated process. Also they are involved in regulating membrane-bound proteins such as ATPase, transport proteins and histocompatibility complexes. In addition, PUFAs regulate expression of some genes, including those coding for fatty-acid synthase, nitric-oxide synthase, sodium-channel proteins. Thus they have an impact on cellular biochemical activities, transport processes and cell-stimulus responses. They are involved in physiological processes including immune responses and cold adaptation, and implicated in pathological conditions such as cardiovascular disease.
Neurons contain a very high percentage of long-chain polyunsaturated fatty acids because they are used to construct complex structures such as the brain, which has very high rates of signal transfer and data processing. Excluding water, the mammalian brain is about 60 percent lipid (lipid is a general term for fatty biochemicals including phospholipids, triglycerides, ceramides and free fatty acids). However the central nervous system is unique compared to other tissues because it cannot directly use alpha-linolenic or linoleic acids, only their long chain PUFA derivates, which are mainly docosahexaenoic acid (DHA) and arachidonic acid (AA) [Broadhurst et al. Br J Nutr 79 (1998) 3-21].
Long chain PUFAs are the building material of the central nervous system and also are required for the normal behavior of cell signaling systems, which determine how neurons function [Clandinin Lipids; 34 (1999) 131-137].
In humans PUFA metabolism and eicosanoid function became important when it was discovered that arachidonate is the precursor for prostaglandins. Ecosanoids are a diverse group of hormones including prostaglandins, thromboxanes and leukotrienes. Research shows that eicosanoid hormones are fundamental to proper maintenance of homeostasis, and are linked to important physiological and pathophysiological conditions. The eicosanoid pathway in mammals begins with the phospholipase-mediated release of PUFAs from membrane phospholipids and is followed by cyclooxygenase-catalysed reactions that give rise to the major classes of metabolites, prostaglandins, thromboxanes, lipoxins and leukotrienes involved in the inflammatory response.
Lately, PUFA chemically related compounds where identified where the alpha amino group of an amino acid forms an amide bond with the carboxylic acid of arachidonic acid. These compounds generically named N-acyl-amino acids include but are not limited to N-arachidonoyl-glycine, N-arachidonoyl-L-serine, N-arachidonoyl-aminobutyric acid [Huang et al. J. B. C. 276 (2001) 42639-42644; Milman et al. PNAS 103 (2006) 2428-2433]. These PUFA derivatives are referred herein as AA-PUFAs for practical reason.
Antinociceptive actions have been described for N-arachidonoylglycine and N-arachidonoylg-aminobutyric and vasodilatory action was associated to N-arachidonoyl L-serine. These AA-PUFAs are also known for their inhibitory properties on fatty acid amide hydrolase. [Cascio et al. BBRC 314 (2004) 192-196; [Huang et al. J. B. C. 276 (2001) 42639-42644; Milman et al. PNAS 103 (2006) 2428-2433].
Interestingly, several arachidonic acid metabolites and other fatty acids have been shown to function as ligands for GPCRs, demonstrating that they can function as mediators, in vivo. Unesterified PUFAs are present in the plasma and in the brain. For example, arachidonic acid and DHA are present at 9 to 22 μM in the plasma and between 3 to 8 nmol/g fresh tissue in the brain, respectively [Kazushige et al. J. Neurochem 63 (1994) 727-736; Rosenberger et al. J. Neurochem 88 (2004) 1168-1178]. N-arachidonoyl-glycine was reported to be present in rat brain at concentration of 50 pmol/g dry tissue [Huang et al. JBC 276 (2001) 42639-42644].
Concentration of Arachidonic acid can be increased by 2 fold after LPS infusion in rat and by 20 fold following ischemia [Cao et al., Life Sciences 78 (2005) 74-81].
PUFAs in Human Nutrition and Disease
The importance of a balanced PUFAs intake has been recognized by health organizations throughout the world over the past decade. There is now some consensus that PUFAs should form a bare minimum 3%, and preferably 10-20%, of the total lipid intake, and that the 6- to 3-ratio should ideally be around 4 or 5:1. Although the biological effects of eicosanoids are undisputed, most diverse pharmacological effects have been proposed for PUFAs. An increase in PUFA consumption carries an elevated risk of exposure to toxic oxidation products, which are implicated in cancer, thrombotic and inflammatory diseases.
A substantial body of evidence links long chain PUFA deficiency to attention-deficit and/or hyperactivity disorders, dyslexia, senile dementia, clinical depression, bipolar disorder, schizophrenia, and other problems of a dual psychological and physiological nature [Peet et al., Marius Press (1999)].
A role of PUFAs was proposed in cocaine addiction [Buydens-Branchey et al., Psychiatry Res. 120 (2003) 29-35], and DHA was shown to ameliorate the impairment of spatial cognition learning ability in amyloid beta-infused rats [Hashimoto et al., J. Nutr. 135 (2005) 549-555]. Specific biological actions of arachidonic acid are described in animal models, such as decrease locomotive activity of mice [Laborit et al., Chem. Biol. Interact. 10 (1975) 309-312], moreover increased arachidonic acid concentration is found in the brain of Flinders Sensitive Line rats, an animal model of depression [Green et al., J. Lipid Res. 46 (2005) 1093-6].
The recognition of such long chain PUFA deficiencies has led many researchers to investigate its connection to numerous psychiatric disorders. So far the correlations have been remarkably positive.
Depression—In the past 100 years, the lifetime risk of developing major clinical depression has increased one hundredfold in North America. This increase coincides with the adoption of a diet based heavily on refined, processed agricultural commodities and a resultant dramatic reduction in n-3 PUFA consumption [Hibbeln & Salem Am. J. Clin. Nutr. 62 (1995) 1-9]. Studies have found that major depression is associated with low blood levels of DHA.
Hyperactivity Disorders and Dyslexia —PUFA deficiency also has been linked to attention deficit-hyperactivity disorder (ADHD) [Stevens et al., Am. J. Clin. Nutr. 62 (1995) 761-768]. Conversion of LA and ALA to long chain PUFA and/or PUFA metabolites in hyperactive children is probably not adequate to maintain normal brain function, or the inadequate conversion exacerbates a preexisting brain abnormality.
In several cases learning and health problems could be associated with low total PUFA levels, especially DHA [Stevens et al., Physiol. Behav. 59 (1996) 915-920].
Dyslexia is often characterized by a visual defect that decreases the eye's ability to adapt to the dark. In a 1995 controlled study conducted in Scotland, supplemental DHA at 480 mg per day for a month was shown to improve this problem in 10 dyslexics [Stordy Dyslexia Rev. 9 (1997) 1-3].
Senile dementia and Alzheimer's disease—Reduced levels of PUFAs have been observed in blood samples from Alzheimer's patients and those suffering from other forms of dementia. Higher levels of fish consumption were correlated to a lower incidence of dementia, including Alzheimer's dementia, in a study of 5,386 Dutch persons over age 55 [Kalmijn et al., Ann. Neurol. 42 (1997) 776-782]. Excessive oxidation of PUFAs in neuronal cell membranes may play a role in the development of Alzheimer's and related dementias.
Schizophrenia and bipolar disorder—Schizophrenia is the most extensively studied neurological disease in relation to lipid metabolism. Red blood cell fatty acids measured in schizophrenics from Ireland, England, Scotland, Japan and the United States have been shown to contain lower than normal levels of AA and DHA, and of PUFAs in general. Schizophrenia may manifest itself when at least two genetic abnormalities in fatty acid metabolism are simultaneously present: an increased rate of removal of PUFAs, especially AA and DHA from phospholipid cell membranes; and a reduced rate of incorporation of these same PUFAs in the cell membranes [Horrobin et al., Schizophr. Res. 30 (1998) 193-208].
Bipolar disorder, alcoholism and schizotypy (antisocial, “disconnected” personality disorder) are also more common in relatives of schizophrenics. Dyslexia and schizotypy arise when only the defect in PUFA incorporation is present [Christensen and Christensen Acta Psychiatr. Scand. 78 (1988) 586-591].